Figure 5

CD4 ⁺ and CD8 ⁺ T cell responses to viral epitopes in TMEV-infected NLM and TLR3KO-SJL mice. (A) Proportions of IFN-γ-producing CD4⁺ cells in the CNS were determined by intracellular staining after stimulation with 2 μM of SJL CD4 capsid (SJL SP) or SJL CD4 noncapsid (SJL NSP) epitope mixtures. Numbers in the FACS plots represent % of IFN-γ producing CD4⁺ cells from total infiltrating CD4⁺ cells. (C) IFN-γ-producing CD4⁺ cell numbers in the CNS at 7 and 21 dpi were shown after stimulation with SJL CD4 capsid (SJL SP) (left panel) or SJL CD4 noncapsid (SJL NSP) (right panel) epitope mixtures. (B) Proportions of IFN-γ-producing CD8⁺ cells in the CNS were determined by intracellular staining after stimulation with 2 μM of SJL CD8 epitope mixture. Numbers in the FACS plots represent % of IFN-γ producing CD8⁺ cells from total infiltrating CD8⁺ cells. Numbers in the bottom panel represent % of H-2K^s-VP3_159-166 tetramer-positive CD8⁺ cells from total infiltrating CD8⁺ cells without further stimulation. (D) IFN-γ-producing CD8⁺ cell numbers after stimulation with SJL CD8 epitopes (left panel) and total numbers of H-2K^s-VP3_159-166 tetramer-reactive CD8⁺ cells in the CNS at 7 and 21 dpi were shown (right panel). (E) The relative expression levels of IFN-γ vs. IL-17 mRNAs in the CNS of virus-infected TLR3KO-SJL and NLM were assessed by real-time PCR. Data are expressed by fold induction after normalization to the GAPDH mRNA levels. The values given are means ± standard deviations of triplicates. Statistically significant differences were indicated with asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (F) Expression of CD69, activation marker of CD4 and CD8 from TMEV-infected NLM and TLR3KO-SJL mice at 7 DPI was analyzed by FACS. Numbers in cytometric plots represent % of positive CD4⁺ or CD8⁺ T cells out of the total CD4⁺ or CD8⁺ T cells, respectively. (G) Expression levels of MHC class I (H-2K^s) and II (I-A^s) molecules on microglia (MG) and macrophages (MP) of TMEV-infected NLM and TLR3KO-SJL mice were analyzed at 7 dpi by flow cytometry. Numbers in cytometric plots represent % of positive MG or MP out of the total MG or MP, respectively.